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The Glucagon-Like Peptide 2 Analog Teduglutide Reversibly Associates to Form Pentamers

      Abstract

      Glucagon-like peptide 1 and 2 and their analog peptide therapeutics are known to reversibly associate to form oligomers. Here we report the association properties of the glucagon-like peptide 2 analog teduglutide at concentrations up to ∼15 mg/mL. Both sedimentation equilibrium (SE-AUC) and sedimentation velocity (SV-AUC) show that teduglutide dissociates completely to monomers below 0.1 mg/mL. SE-AUC shows that the apparent weight-average molar mass increases substantially between 0.1 and 1 mg/mL, reaching a maximum of ∼14.5 kDa (∼3.9-mer) near 2 mg/mL, and then falling at higher concentrations because of strong solution nonideality effects (highly positive second virial coefficient). Circular dichroism spectra over the range from 0.1 to 2 mg/mL show that self-association is accompanied by significant increases in alpha-helix content, and that the associated state has a distinct tertiary structure. The SV-AUC data up to 2.2 mg/mL are fitted fairly well by an ideal rapidly reversible monomer-pentamer association. The SE-AUC modeling included thermodynamic nonideality effects. SE-AUC data up to ∼15 mg/mL imply a monomer-pentamer association at lower concentrations, but the pentamers also appear to weakly associate to form decamers. These results illustrate the importance of directly modeling the solution nonideality effects, which if neglected would lead to an incorrect preferred stoichiometry.

      Keywords

      Abbreviations used:

      CD (circular dichroism), DLS (dynamic light scattering), DPBS (Dulbecco’s phosphate-buffered saline), GLP-1 (glucagon-like peptide 1), GLP-2 (glucagon-like peptide 2), NMR (nuclear magnetic resonance), SAXS (small-angle X-ray scattering), SE-AUC (sedimentation equilibrium analytical ultracentrifugation), SEC-MALS (size-exclusion chromatography with multi-angle static light scattering detection), SV-AUC (sedimentation velocity analytical ultracentrifugation), TFE (trifluoroethanol), UV (ultraviolet)
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